Mucus forms an essential barrier that protects the lungs from inhaled particles, pathogens and chemicals. These toxicants are entrapped in mucus, then swept out of the lungs by ciliary action. Paradoxically however, mucus dysfunction contributes to the pathobiology of all of the common diseases of the airways, including asthma, cystic fibrosis and COPD, as well as to interstitial lung diseases. Mucin glycoproteins are the principal macromolecular component of mucus, responsible for its structure as a semi-solid gel by interacting with several hundred-fold their mass of water. Mucins are secreted both at a low baseline rate and a high stimulated rate. A common feature of airway mucus dysfunction is the rapid secretion of hyperproduced mucins into the airway lumen, forming mucus that is excessively viscoelastic and cannot be cleared by ciliary action. This impedes airflow and provides a protected environment for microbial growth While the control of mucin production and hydration have been studied intensively, the mechanism of secretion is incompletely understood. Exocytosis in all eukaryotes is mediated by the cooperative interactions of a SNARE complex and an SM scaffolding protein, which are the core exocytic machinery. Our studies show that for some components of this machinery, different isoforms function in basal versus stimulated mucin secretion. Our central hypothesis is that two different VAMP proteins define two distinct classes of mucin secretory granules, associated with distinct secretory function and with distinct trafficking regulatory proteins. Aim 1. Determine the roles of VAMP3 and VAMP8 in defining two distinct structural and functional classes of mucin secretory granules. Aim 2. Determine how mucin granules are assembled, from their exit from the trans-Golgi network, through homotypic fusion, to form two classes of mature fusion-ready granules. Aim 3. Determine the physiological and pathophysiological consequences of manipulating the traffic of the two mucin granule classes. Completion of these aims will provide fundamental understanding of the mucin secretory mechanism, and will apply that knowledge to test translational strategies for mitigating mucus dysfunction while preserving its protective benefits.